The polarization of light emitted by an appropriately excited fluorescent solution can be defined by the intensity of two linearly polarized components, one vibrating along an axis orthogonal to the plane defined by the axes of the excitation light and the emitted light, and the other vibrating along an axis in that plane and orthogonal to the first component. If the first and second component intensities are respectively designated I.sub..perp. and I.sub..parallel., the polarization of the fluorescent emission is ##EQU1##
The measurement of the polarization of fluorescence is useful in the study of immune reactions and in biological assay and is also more generally useful for molecular analysis. For example, the polarization of fluorescence is of interest in the detection of change in molecular shape such as in protein denaturation, polymerization studies, monitoring of oil cracking reactions, and food process monitoring.
The following articles describe the measurement and some applications of polarization of fluorescence and apparatus for such measurement: G. Weber, "Photoelectric Method for the Measurement of the Polarization of the Fluorescence of Solutions," Journal of the Optical Society of America, Vol. 46, No. II, November 1956, pages 962-970; D. A. Deranleau, "A Recording Fluorescence Polarization Photometer," Analytical Biochemistry, Vol. 16, 1966, pages 438-449; R. J. Kelly, W. B. Dandliker and D. E. Williamson, "Digital, Photon-Counting Fluorescence Polarometer," Analytical Chemistry, Vol. 48, No. 6, May 1976, pages 846-856; R. D. Spencer, F. B. Toledo, B. T. Williams and N. L. Yoss, "Design, Construction, and Two Applications for an Automated Flow-Cell Polarization Fluorometer with Digital Read Out: Enzyme-Inhibitor (Antitrypsin) Assay and Antigen-Antibody (Insulin-Insulin Antiserum) Assay," Clinical Chemistry, Vol. 19, No. 8, 1973, pages 838-844; P. Johnson and E. G. Richards, "A Simple Instrument for Studying the Polarization of Fluorescence," Archives of Biochemistry and Biophysics, Vol. 97, 1962, pages 250- 259; S. Ainsworth and E. Winter, "An Automatic Recording Polarization Spectrofluorimeter," Applied Optics, Vol. 3, No. 3, March 1964, pages 371-383; R. F. Chen and R. C. Bowman, "Fluorescence Polarization: Measurement with Ultraviolet - Polarizing Filters in a Spectrophotofluorometer," Science Vol. 147, 1965, pages 724-731.
In a known system for polarization of fluorescence measurement, a polarizer is aligned first along an axis parallel to the polarization axis of the excitation light and next along an axis orthogonal to the polarization axis of the excitation light, and orthogonal to the first axis, a measurement of the intensity of emitted fluorescent light being made for each polarizer position. A computation is then made of the sum and difference components and the ratio therebetween according to the above equation. A polarizer can be employed which is moved between two mutually orthogonal positions, as shown in the above-identified article of Chen and Bowman, or continuously rotated, as shown in the article of Kelly, Dandliker and Williamson. Alternatively, plural optical channels have been employed, as shown in Weber, each channel having polarized light respectively polarized in one of the two orthogonal positions. The vertically and horizontally polarized light is received by either a single photomultiplier tube or a separate photomultiplier tube for each channel. Often, an additional photomultiplier is employed to monitor the intensity of the excitation source for use in an automatic gain control circuit.
The system implementations known in the art have been quite complex both with respect to the optical arrangements employed and the electronic circuitry for computing polarization. Computation of the polarization equation has in general been accomplished by analog or digital computer or specific ratio determining apparatus such as a ratio recorder.